Bioactive peptides having detrimental effects on pest slugs

ABSTRACT

Provided herein are synthetic peptides developed from slug and insect neuropeptides. Peptides described can be used to repel, control or deter slugs, including the gray garden slug ( Deroceras reticulatum ), from feeding on agricultural and horticultural plants. The peptides can be combined with bait materials, or applied directly to plants or areas, or produced by genetically modified plants.

CROSS-REFERENCE

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 62/892,605 filed Aug. 28, 2019, the content ofwhich is expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of Invention

Provided herein are synthetic peptides developed from slug and insectneuropeptides. Peptides described can be used to repel, control or deterslugs, including the gray garden slug (Deroceras reticulatum), fromfeeding on agricultural and horticultural plants. The peptides can becombined with bait materials, or applied directly to plants or areas.

Background

The gray garden slug, or gray field slug, Deroceras reticulatum, isnative to Europe, omnivorous, and is the most destructive pest on avariety of crops in greenhouses and fields. Infestations and theresulting damage/problems associated with slugs are increasingworldwide. Slugs affect a wide range of cropping systems including seedproduction, field crops, row crops, Christmas tree farms, andhorticultural nurseries. In Oregon alone, in new fields, the cost ofbait applications, direct loss of plants, and the cost of replanting isestimated to cost the seed industry >$50 million annually. Inestablished grass seed fields, slug bait applications are estimated at˜$7 million annually, with an additional $38 million in losses from cropdamage. Globally, those economic impacts are increasing every year.

Currently, major control methods for slugs rely on conventionalpesticides. Multiple applications of sprays, baits or granules are usedregardless of the level of residues accumulating in a field.Unfortunately, some of these chemicals are non-specific, and killnon-target and beneficial organisms such as earthworms in the soil.Additionally, this strategy is costly and inefficient at suppressingslugs to a level below a certain economical threshold becauseappropriate soil moisture and weather conditions are necessary foreffective delivery of the chemical and consequent slug control in thefield. Another risk of chemical applications is the potential fordeveloping chemical resistance in slugs (Salmijah et al, Plant Prot.Quart., (2000) 15:2-5. As presented herein, we have developed novelpeptides that are commercially viable and specific for slugs.

SUMMARY OF THE INVENTION

In one embodiment, the present disclosure provides a synthetic peptidehaving SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 4, or acombination thereof. Such compositions can include any one of thesesequences specifically, namely, SEQ ID NO: 18, SEQ ID NO: 17, SEQ ID NO:3, or SEQ ID NO: 4.

Another embodiment provided herein is a molluscicide for controlling aslug, the composition comprising an effective amount of a syntheticpeptide having SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 4,or a combination thereof such compositions can include any one of thesesequences specifically, namely, SEQ ID NO: 18, SEQ ID NO: 17, SEQ ID NO:3, or SEQ ID NO: 4. In some embodiments, the molluscicide also has anagriculturally acceptable carrier. In other embodiments, molluscicidesdisclosed herein can also have a preservative, a dispersant, afungicide, an herbicide, an attractant, a phagostimulant, a baitmaterial, or a combination thereof.

Another embodiment disclosed herein is a bait composition forcontrolling a slug, comprising: an effective amount of a syntheticpeptide having SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 4,or a combination thereof, a food material; and optionally aphagostimulant.

The present disclosure further provides a method for controlling a slugcomprising contacting a slug or its environment with a biologicallyeffective amount of a synthetic peptide or molluscicide, wherein themortality of said slug increases. In specific embodiments, the slug isDeroceras reticulatum.

An additional embodiment disclosed herein is a composition comprising asynthetic peptide having SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or acombination thereof.

Also provided herein is a molluscicide composition for controlling aslug, the composition comprising an effective amount of a syntheticpeptide having SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a combinationthereof.

INCORPORATION BY REFERENCE

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims. Features and advantages of the present invention arereferred to in the following detailed description, and the accompanyingdrawings of which:

FIG. 1 provides a graphic representation of peptide ligand screeningresults. Peptides described herein (Table 1) produced fluorescencesignals when exposed to recombinant Sf9 cells expressing receptorvariant A or variant B. Unmodified Sf9 cells were tested as a negativecontrol.

FIG. 2A and FIG. 2B provide a graphic representation of the dosageresponse of two peptides: SEQ ID NO: 3 (FIG. 2A) and SEQ ID NO: 4 (FIG.2B) on two receptors (variant A or variant B.)

FIG. 3 provides a graphic representation of phenotypic effects ofinjection of molluscicidal peptides. Body weight loss induced byinjecting gray garden slugs with water control (left column), SEQ ID NO:3 (middle column), or SEQ ID NO: 4 (right column).

FIG. 4 provides a pictorial representation of altered mucus productionin untreated (panel A) and treated (panel B) slugs. Slugs were injectedwith water only (panel A) or—SEQ ID NO: 3 (panel B).

FIG. 5 provides a graphic representation of the dosage response ofseveral synthetic peptides on two GPCRs (variant A or variant B.)

FIG. 6 and FIG. 7 : provides a graphic representation of phenotypiceffects of injection of molluscicidal peptides.

FIG. 6 provides a graphic representation of phenotypic effects ofinjection of molluscicidal peptides. Mortality of slugs injected withSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 25, or SEQ ID NO:26 within 24 h under dry conditions (Petri dish) is shown.

FIG. 7 provides a graphic representation of phenotypic effects ofinjection of molluscicidal peptides. Mortality of slugs injected withSEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 25 or SEQ ID NO: 26 within 24 hunder wet conditions (Petri dish with wet paper towel) is shown.

FIG. 8 provides a pictorial representation of altered mucus productionin slugs fed on lettuce treated water only (panel A) and lettuce treatedwith SEQ ID NO: 3 (panel B).

FIG. 9A, FIG. 9B and FIG. 9C provide a graphic representation ofanti-feeding effects of peptides. Slugs were fed with lettuce treatedwith water or—lettuce treated with SEQ ID NO: 19, SEQ ID NO: 25, or SEQID NO: 26 and data was taken within 1 h (FIG. 9A), 2 h (FIG. 9B) or 6 h(FIG. 9C).

DETAILED DESCRIPTION OF THE INVENTION

Neuropeptides are part of a large group of neurohormones that haveimportant regulatory functions and are found in invertebrates. A varietyof peptide families from Mollusca have been identified and classified bytheir core structures and functionalities. These neuropeptide ligandsbind to G Protein-coupled receptors (GPCRs), a large group of signalingreceptors for various signal transductions. GPCRs are membrane embeddedproteins, also known as 7 transmembrane receptors, activated by a widevariety of stimulants including light, odorant molecules, peptide andnon-peptide neurotransmitters, hormones, growth factors and lipids. Theycontrol a wide variety of physiological processes including sensorytransduction, cell-cell communication, neuronal transmission, andhormonal signaling.

The PRXamide (X=any amino acid) family of neuropeptides is based on thecore amino acid sequence at the C-terminal end that are required foractivity and on sequence homology of their GPCRs (Jurenka, R., Adv.Insect Physiol., (2015) 49: 123-70). The PRXamide family ofneuropeptides are ubiquitous in invertebrate animals. The familyincludes proteins such as pyrokinin, pheromone biosynthesis-activatingneuropeptide, diapause hormone, CAPA/periviscerokinin (a.k.a.cardioacceleratory peptide 2b), and ecdysis triggering hormone in manyarthropods and gastropods. However, knowledge about structure ofspecific peptide ligands and their receptors is necessary to more fullyunderstand their interactions to facilitate the development ofantagonists and agonists.

Disclosed herein are specific synthetic peptides that target slug GPCRs,interfering with normal functioning and leading to detrimental effects.In specific embodiments, the peptides (SEQ ID NO: 3; SEQ ID NO: 4; SEQID NO: 19; SEQ ID NO: 25; SEQ ID NO: 26) provided a novel approach forbio-control of these important agricultural and horticultural pests.

Preferred embodiments of the present invention are shown and describedherein. It will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will occur to those skilled in the artwithout departing from the invention. Various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is intended that the included claims definethe scope of the invention and that methods and structures within thescope of these claims and their equivalents are covered thereby.

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the instantinvention pertains, unless otherwise defined. Reference is made hereinto various materials and methodologies known to those of skill in theart. Standard reference works setting forth the general principles ofrecombinant DNA technology include Sambrook et al., “Molecular Cloning:A Laboratory Manual”, 2^(nd) ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y., 1989; Kaufman et al., eds., “Handbook of Molecular andCellular Methods in Biology and Medicine”, CRC Press, Boca Raton, 1995;and McPherson, ed., “Directed Mutagenesis: A Practical Approach”, IRLPress, Oxford, 1991.

Any suitable materials and/or methods known to those of skill can beutilized in carrying out the instant invention. Materials and/or methodsfor practicing the instant invention are described. Materials, reagentsand the like to which reference is made in the following description andexamples are obtainable from commercial sources, unless otherwise noted.

As used in the specification and claims, use of the singular “a”, “an”,and “the” include plural references unless the context clearly dictatesotherwise.

The terms isolated, purified, or biologically pure as used herein, referto material that is substantially or essentially free from componentsthat normally accompany the referenced material in its native state.

The term “about” is defined as plus or minus ten percent of a recitedvalue. For example, about 1.0 g means 0.9 g to 1.1 g and all valueswithin that range, whether specifically stated or not.

The term “a nucleic acid consisting essentially of”, and grammaticalvariations thereof, means nucleic acids that differ from a referencenucleic acid sequence by 20 or fewer nucleic acid residues and alsoperform the function of the reference nucleic acid sequence. Suchvariants include sequences which are shorter or longer than thereference nucleic acid sequence, have different residues at particularpositions, or a combination thereof.

“Activity” of a synthetic peptide, as used herein, refers to thecapacity to obtain mortality or paralysis in slugs when such slugs areexposed to the peptides (e.g., via feeding or injection), whichmortality or paralysis is significantly higher than a negative control(e.g., a buffer).

“Carrier” as used herein refers to any method of dispersal,dispensation, application, timed-release, encapsulation,microencapsulation, or the like to apply the slug-affecting compositionas further described herein. In embodiments, such “carriers” may includea variety of microencapsulation, controlled-release, and otherdispersion technologies available to those of ordinary skill in the art.

“Control” or “controlling” as used herein refers to any means forpreventing infestation, reducing the population of already infestedareas, or elimination of pest population(s) whose “control” is desired.Indeed, “controlling” as used herein refers to any indicia of success inprevention, elimination, reduction, repulsion, or amelioration of a pestpopulation or pest problem.

An “effective amount” is an amount sufficient to effect desiredbeneficial or deleterious results. In terms of treatment, an “effectiveamount” is that amount sufficient to make the target pest non-functionalby causing an adverse effect on that pest, including (but not limitedto) physiological damage to the pest; inhibition or modulation of pestgrowth; inhibition or modulation of pest reproduction; or death of thepest. The exact amount required can vary from composition to compositionand from function to function, depending on recognized variables such asthe compositions and processes involved. An effective amount can bedelivered in one or more applications. Thus, it is not possible tospecify an exact amount, however, an appropriate “effective amount” canbe determined by the skilled artisan via routine experimentation.

The terms “polypeptide, peptide or protein” refer to polymers in whichthe monomers are amino acid residues which are joined together throughamide bonds. When the amino acids are alpha-amino acids, either theL-optical isomer or the D-optical isomer can be used. The terms are usedinterchangeably herein. These terms apply to amino acid polymers inwhich one or more amino acid residues are an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers.

A “conservative substitution” in a polypeptide is a substitution of oneamino acid residue in a protein sequence for a different amino acidresidue having similar biochemical properties. Typically, conservativesubstitutions have little to no impact on the activity of a resultingpolypeptide. For example, a protein or peptide including one or moreconservative substitutions (for example no more than 1, 2, 3, 4 or 5substitutions) retains the structure and function of the wild-typeprotein or peptide. A polypeptide can be produced to contain one or moreconservative substitutions by manipulating the nucleotide sequence thatencodes that polypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR. In one example, such variants can bereadily selected by testing antibody cross-reactivity or its ability toinduce an immune response. Conservative substitutions generally maintain(a) the structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b) thecharge or hydrophobicity of the molecule at the target site, or (c) thebulk of the side chain. The substitutions which in general are expectedto produce the greatest changes in protein properties will benon-conservative, for instance changes in which (a) a hydrophilicresidue, for example, serine or threonine, is substituted for (or by) ahydrophobic residue, for example, leucine, isoleucine, phenylalanine,valine or alanine; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,for example, lysine, arginine, or histidine, is substituted for (or by)an electronegative residue, for example, glutamine or aspartate; or (d)a residue having a bulky side chain, for example, phenylalanine, issubstituted for (or by) one not having a side chain, for example,glycine.

The term “phagostimulant” refers to any substance that will entice theslug to ingest the selected bioactive peptide. Suitable phagostimulantsinclude but are not limited to syrups, honey, aqueous solutions ofsucrose, artificial sweeteners such as sucralose, saccharin, and otherartificial sweeteners, starch, amino acids, and other proteins.Additionally, the bait material containing the bioactive peptidedisclosed herein would be incorporated in water soluble baits, oil-inwater or oil/water emulsion baits, liquid type or gel type of baits.

The ready-to-use preparations of phagostimulants can be in the form of awettable powder, flowable concentrate solution, water soluble granules,ultra-low volume formulation, and the like, which can be applied to thetarget habitat. Phagostimulants can be used in combination with peptidesof the present disclosure to enhance or encourage uptake by targetpests. In essence, the combination is a slug bait. Such baits can alsoinclude any other component desired by one of skill in the art, such ascarriers, preservatives, odorants, molluscicides, insecticides and thelike. Phagostimulants can include carbohydrates such as glucose,fructose, arabinose, sorbitol, maltose, glucose, lactose, or any othersmall sugar. It will be obvious to a person skilled in the art that somecarbohydrates and/or amino acids are likely to act as a deterrent. Thus,a bait, or other composition of the present invention can includephagostimulant(s) that attract a target slug and components that repelother animals (such as beneficial insects, pets and wildlife). Suchvariations are easily appreciated by any person skilled in the art.

The “sequence identity” of two related nucleotide or amino acidsequences, expressed as a percentage, refers to the number of positionsin the two optimally aligned sequences which have identical residues(×100) divided by the number of positions compared. A gap, i.e., aposition in an alignment where a residue is present in one sequence butnot in the other is regarded as a position with non-identical residues.The alignment of the two sequences is performed by the Needleman-Wunschalgorithm (Needleman and Wunsch, J Mol Biol, (1970) 48:3, 443-53). Acomputer-assisted sequence alignment can be conveniently performed usinga standard software program such as GAP which is part of the WisconsinPackage Version 10.1 (Genetics Computer Group, Madison, Wis., USA) usingthe default scoring matrix with a gap creation penalty of 50 and a gapextension penalty of 3.

Peptides

The peptides provided herein can be synthesized by any suitable method,such as exclusively solid-phase techniques, partial solid-phasetechniques, fragment condensation, or classical solution addition. Theamino acids of the compounds of the invention are typically joined toadjacent groups through amide linkages. For example, without beinglimited thereto, the peptide variants may be synthesized by methods wellknown to those skilled in the art of peptide synthesis, e.g., solutionphase synthesis [see Finn and Hoffman, In “Proteins,” Vol. 2, 3rd Ed.,H. Neurath and R. L. Hill (eds.), Academic Press, New York, pp. 105-253(1976)], or solid phase synthesis [see Barany and Merrifield, In “ThePeptides,” Vol. 2, E. Gross and J. Meienhofer (eds.), Academic Press,New York, pp. 3-284 (1979)], or stepwise solid phase synthesis asreported by Merrifield [J. Am. Chem. Soc. 85: 2149-2154 (1963)], thecontents of each of which are incorporated herein by reference. However,the peptide fragments are preferably produced by recombinant DNAtechniques, which are particularly suitable for large-scale use.

Synthesis by the use of recombinant DNA techniques, for the purpose ofthis application, should be understood to include the suitableemployment of structural genes coding for the sequence as specifiedhereinafter. The synthetic peptides may also be obtained by transforminga microorganism or plant using an expression vector including a promoteror operator, or both, together with such structural genes and causingsuch transformed microorganisms or plant to express the peptide.

Vectors used in practicing the present invention are selected to beoperable as cloning vectors or expression vectors in the selected hostcell. Numerous vectors are known to practitioners skilled in the art,and selection of an appropriate vector and host cell is a matter ofchoice. The vectors may, for example, be bacteriophage, plasmids,viruses, or hybrids thereof, such as those described in Sambrook et al.[Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory,1989] or Ausubel et al. [Current Protocols in Molecular Biology, JohnWiley & Sons, Inc, 1995], the contents of each of which are hereinincorporated by reference. Further, the vectors may be non-fusionvectors (i.e., those producing the peptides of the invention not fusedto any heterologous polypeptide), or alternatively, fusion vectors(i.e., those producing the peptides fused to a vector encodedpolypeptide). The fusion proteins would of course vary with theparticular vector chosen.

TABLE 1 Synthetic peptide sequences SEQ ID Sequence Source NO: PMSMLRLD. reticulatum SEQ ID NO: 1 GQFSAARL D. reticulatum SEQ ID NO: 2 QPPLPRYD. reticulatum SEQ ID NO: 3 QPPVPRY D. reticulatum SEQ ID NO: 4FFFRPAPRG D. reticulatum SEQ ID NO: 5 VFYTKSDDNDYPRI D. reticulatumSEQ ID NO: 6 GIFTQSAHGYSPRV D. reticulatum SEQ ID NO: 7 SGYLAFPRMD. reticulatum SEQ ID NO: 8 TGPSASSGLWFGPRL D. melanogatser SEQ ID NO: 9SVPFKPRL D. melanogatser SEQ ID NO: 10 DDSSPGFFL D. melanogatser SEQ IDKITKNVPRL NO: 11 LSDDMPATPADQEMYRQ H. zea SEQ ID DPEQIDSRTKYFSPRL NO: 12FYAPFSPRL H. halys SEQ ID NO: 13 DAGLFPFPRV H. halys SEQ ID NO: 14EQLIPFPRV H. halys SEQ ID NO: 15 NGASGNGGLWFGPRL H. halys SEQ ID NO: 16QPPXPRY Generic SEQ ID  formula NO: 17 XXXXPRY Generic SEQ ID formulaNO: 18 APPLPRY Synthetic SEQ ID NO: 19 QAPLPRY Synthetic SEQ ID NO: 20QPALPRY Synthetic SEQ ID NO: 21 QPPAPRY Synthetic SEQ ID NO: 22 QPPLARYSynthetic SEQ ID NO: 23 QPPLPAY Synthetic SEQ ID NO: 24 QPPLPRASynthetic SEQ ID NO: 25 LPRY Synthetic SEQ ID NO: 26 PPLPRY SyntheticSEQ ID NO: 27 PLPRY Synthetic SEQ ID NO: 28

Peptide Compositions

In particular embodiments, the present invention provides a compositionhaving synthetic peptide represented by one or more of SEQ ID NOs: 1-28(Table 1). The synthetic peptides were developed based on sequences fromDeroceras reticulatum (gray garden slug) and various insects (Drosophilamelanogaster, Helicoverpa zea, and Halyomorpha halys). In specificembodiments, compositions containing peptides having SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 27, SEQ ID NO: 28, or a combination of two or more of thesepeptides is provided. Peptides utilized herein, can be any one withinthe generic formulas provided (SEQ ID NO: 17, SEQ ID NO: 18). Typically,peptides of the present disclosure are provided to a target recipient(e.g., slug) in an amount sufficient to induce a detrimental effect(e.g., death). Peptides of the present disclosure can be in the amideform—having an (NH₂) group at the C-terminus.

Typically, a peptide or peptide mixture of the present invention isprovided to a target pest in an amount sufficient to cause a detrimentaleffect (e.g., paralysis or death) to a mollusk, such as a slug or snail.For example, when a slug is feeding on peptide-laden plant material(e.g., leaf), the slug ingests a sufficient level of peptide to resultin a detrimental effect. In some embodiments, a combination of two ormore peptides (e.g., a combination of SEQ ID NO: 3 and SEQ ID NO: 4) canbe combined in a single treatment, on a single plant, or applied to atarget area. Where two or more peptides are utilized, they can beprovided in a single application, or in multiple, sequentially appliedapplications.

In addition, the peptide(s) of the present invention, compositions ofthe present invention that are intended to be applied to a plant, or anarea, can also comprise one or more chemoattractants, phagostimulants,visual attractants, insecticides, pheromones, fungicides, orcombinations thereof. Such additional components are well known in theart and are readily chosen to complement compositions of the presentinvention, but are not specifically integral to the present invention.These additional components can be formulated to be coated on a plant,plant part, leaf, fruit, vegetable, stem or other plant structure. Incertain aspects the additional component(s) are combined with one ormore excipients, buffering agents, carriers, etc. that are also wellknown in the art.

In some embodiments of the present disclosure, one or more peptidesprovided herein are expressed in a transgenic plant, such that ingestionof the transgenic plant tissues by a target pest leads to the effect.Methods of producing transgenic plants are well known in the art.

Application to Target Plants

Compositions of the inventions disclosed herein can be applied to soil,fruits, vegetables, crops, and any other desired target using anydelivery methodology known to those of skill in the art. For example,peptide-containing compositions can be applied to the desired locale viamethods and forms including, but not limited to, sprays, granules,flood/furrow methods, sprinklers, fumigation and drip irrigation. Inembodiments of the disclosure where the compositions are sprayed onto adesired locale, the compositions can be delivered as a liquidsuspension, emulsion, microemulsion or powder. In other embodiments,granules or microcapsules can be used to deliver the compositions of thedisclosure.

The compositions of the present disclosure can be applied to plantsand/or crops by any convenient method, for example, by using a fixedapplication system such as a center pivot irrigation system. Applicationto fields of plants and/or crops is made by air spraying, i.e., from anairplane or helicopter, or by land spraying. For example, land sprayingmay be carried out by using a high flotation applicator equipped with aboom, by a back-pack sprayer or by nurse trucks or tanks. One of skillin the art will recognize that these application methodologies areprovided by way of example and that any applicable methods known in theart or developed in the future can be utilized.

Having generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedherein to further illustrate the invention and are not intended to limitthe scope of the invention as defined by the claims.

EXAMPLES Example 1

Slugs

The gray garden slugs utilized herein (Deroceras reticulatum) werecollected in Corvallis, Oreg., USA, and maintained with carrot andlettuce in a controlled incubator (13° C., 90% RH, 14:10=light:dark, dimlight).

RNA Isolation and cDNA Synthesis

Mature slugs were individually homogenized after removing the surfaceslime in lysis buffer (PURELINK RNA Mini Kit—Invitrogen, Thermo FisherScientific, Waltham, Mass., USA) using a PYREX® glass pestle tissuegrinder (Corning, Corning, N.Y., USA). Total RNA was isolated using thePURELINK RNA Mini Kit according to manufacturer's instructions. Thetotal RNA was quantified by NanoDrop 2000 spectrophotometer (ThermoFisher Scientific) and stored at −80° C. until use. For 5′- and 3′-rapidamplification of cDNA ends (RACEs)-PCR, 5′- and 3′-RACE-ready cDNAs weresynthesized with 1 μg of total RNA using SMARTer® RACE 5′/3′ Kit(Clontech Laboratories, Takara, Mountain View, Calif., USA). Thefirst-strand cDNA was synthesized with 1 μg of total RNA using aSuperScript IV First-Strand Synthesis System (Invitrogen). cDNAs werestored at −20° C. until use.

Molecular Cloning of the Slug Receptor A and B

Both 5′- and 3′-RACEs of the partial transcript sequences obtained fromthe slug transcriptome were amplified using Phusion High Fidelity DNAPolymerase (Thermo Fisher Scientific) under the following PCRconditions: 98° C. for 30 s, 35 cycles of 98° C. for 10 s, 68° C. for 20s, and 72° C. for 1 min, then 72° C. for 10 min using a Veriti 96 FastThermal Cycler (Applied Biosystems, Foster City, Calif., USA). Using apair of gene-specific primers, target sequences were amplified from thefirst-strand cDNA using Phusion High Fidelity DNA polymerase under thefollowing PCR conditions: 98° C. for 30 s, 35 cycles of 98° C. for 10 s,55° C. for 20 s, and 72° C. for 1 min, then 72° C. for 10 min usingVeriti 96 Fast Thermal Cycler. PCR products were run in 1.2% agarosegel, purified and cloned into pJET1.2 vector (Thermo Fisher Scientific)for sequencing. The sequencing results were analyzed using Geneious 8.1software (Biomatters, Newark, N.J., USA).

Functional Expression of the Slug Receptor A and B

The open reading frames (ORFs) of D. reticulatum GPCRs were insertedinto a pIB/V5-His TOPO® TA expression vector (Thermo Fisher Scientific)to be expressed in Sf9 cells as described previously (Choi et al., Gen.Comp. Endocrinol., (2017) 246:354-62). To add a Kozak sequence(G/ANNATGG) in the translation initiation the ORFs of D. reticulatumGPCRs were amplified using specific primer sets. The sequences of twodifferent ORFs of D. reticulatum GPCRs were confirmed after insertioninto the expression vector.

Binding Assay with Peptides

Small synthetic peptides derived from the PRXamide family membersidentified from the gray garden slug and insects (Table 1), were testedto determine their binding activities to two slug GPCRs (receptor A andB) expressed in the Sf9 insect cell line. All peptide ligands of SEQ IDNOs: 1-16 and 19-28, as well as the “RY” dipeptide and “PRY” tripeptide(purity>95%) were synthesized from Peptide 2.0 (Chantily, Va., USA). Sf9cells were cultured as described previously (Choi et al., supra). About50,000 cells per well in a 96-well plate (Corning C3603) were incubatedat 28° C. overnight. At 48 h, cells were rinsed with 100 μL of freshmedium without fetal bovine serum (FBS). Then, cells were incubated in95 μL of 1×FLIPR Calcium 6 reagent (Molecular Devices, San Jose, Calif.,USA) containing 2.5 mM probenecid at room temperature in the dark for 1h. The reagent-loaded cells were transferred to the Flexstation 3multimode microplate reader (Molecular Devices) equipped with filters(excitation: 485 nm and emission: 520 nm) and an 8-channel pipettor.Fluorescence measurements from each well on the column were taken every5 s for 4 min. After 30 s the peptide ligand (10 μL) was added with thepipettor and fluorescent intensity was measured for up to 3 min. Then, 5μL of 1 μM ionomycin (Thermo Fisher Scientific) was added to the cellsto obtain a maximum fluorescence reading. Baseline fluorescence wasdetermined by averaging 5 time points from each well prior to treatmentwith ligand and the resulting response was expressed as a percentincrease in fluorescence relative to baseline value. Cells were testedonly once with a ligand then discarded. Data were analyzed usingMicrosoft Excel as described previously, and half-maximal effectiveconcentration (EC₅₀) values of ligands were determined using GraphPadPrism 6 (GraphPad Software, Inc., San Diego, Calif.).

Results of the initial screening of each of the peptides on Sf9 cells,indicated that two of the peptides were the most likely candidates foradditional analysis (FIG. 1 ). The binding affinity to the two receptorsof these two peptides, QPPLPRY-NH₂ (SEQ ID NO: 3) and QPPVPRY-NH₂(SEQ IDNO: 4), was measured based on specific fluorescent intensity of the Sf9cells expressing receptor A or receptor B in a 96-well plate. A strongfluorescent signal indicated strong binding activity between the peptideligand and receptor. The EC₅₀ values of SEQ ID NO: 3 (FIG. 2A) and SEQID NO: 4 (FIG. 2B) peptides were 1.4 and 12.7 nM, respectively, asdetermined with in vitro cell line experiments.

Example 2

Body Weight Loss Induced by Peptides

To test their effects on slugs, peptides of SEQ ID NO: 3 and SEQ ID NO:4 were dissolved in 5 μl of purified water (10 nmol) and injected intofour gray garden slugs per each treatment. Over 80% of the slug bodyweight was lost after 72 h when either peptide was injected into theslugs, almost 2-fold higher than the water control (FIG. 3 ).Additionally, slugs injected with peptide (SEQ ID NO: 3) excreted largevolumes of milky mucus within 1 minute, exhibited body paralysis,extruding tentacles, weight loss and dehydration (FIG. 4 , exemplaryresults).

Example 3

Mortality Induced by Peptides

To test the effect of varying individual amino acid residues and varyingthe length of the peptide of SEQ ID NO: 3, different amino acid residueswere replaced with alanine (A), or the overall length of the peptide wasreduced by sequentially removing amino acid residues from the N-terminalend of SEQ ID NO: 3 (Table 1, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 26, SEQ ID NO: 27, SEQ ID NO: 28). Altered peptides were tested andmeasured for binding activity with the receptors using the same methodsas described above. Some altered peptides (SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22) maintained binding activity and activatedthe receptors (FIG. 5 , exemplary results). Reduced peptides (SEQ ID NO:26, SEQ ID NO: 27, SEQ ID NO: 28) also retained the ability to activatethe receptors (FIG. 5 , exemplary result). The results in these two casestudies indicated that the RY and PRY sequences located at the C-terminiof peptides may play a role in the binding activity of these peptides.These results suggest that similar small peptides also can be alsomodified and/or conjugated with other peptides or compounds to activatereceptors.

To test their effects on slugs, 100 nmol of each of the peptides (SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 25 and SEQ ID NO: 26) wasdissolved in 5 μL of purified water (100 nmol) and injected into 10 graygarden slugs per each treatment. Under dry conditions, after 24 h theslug mortalities observed were 40% with SEQ ID NO: 1, 70% with SEQ IDNO: 3, 90% with SEQ ID NO: 19, 70% with SEQ ID NO: 25, and 70% with SEQID NO: 26. However, no mortalities were observed in the control (waterinjected) slugs (FIG. 6 , exemplary result). Under wet conditions, theslug mortalities observed within 24 h were 40% with SEQ ID NO: 19, 40%with SEQ ID NO: 25, and 30% with SEQ ID NO: 26. No mortalities wereobserved in the control (water injected) or in SEQ ID NO: 3 injectedslugs (FIG. 7 , exemplary result).

Example 4

Anti-Feeding Induced by Peptides

To test for the effect of the peptides on feeding, slugs were fed a dietof lettuce leaf tissues treated with water alone or with 200 nmol ofindividual peptides (SEQ ID NO: 19, SEQ ID NO: 25, and SEQ ID NO: 26)dissolved in 10 μL water (FIG. 8 ). Ten gray garden slugs were used foreach treatment and each treatment was replicated three times.

The maximum anti-feeding effect was observed with SEQ ID NO: 19.Anti-feeding was significantly (p<0.05) increased at 93.3% within 1 hwith SEQ ID NO: 19 compared to the water treatment (control) value of48.0%. Peptides SEQ ID NO: 25 (66.7%) and SEQ ID NO: 26 (73.3%) did notshow statistically significant anti-feeding effects (FIG. 9A, exemplaryresult). The maximum anti-feeding effect of SEQ ID NO: 19 on slugs wassignificantly (p<0.01) increased at 91.3% within 2 h when compared tothe water treatment (13.3%), but peptides SEQ ID NO: 25 (46.7%) and SEQID NO: 26 (48.0%) did not show statistically significant anti-feedingeffects (FIG. 9B, exemplary result). The maximum anti-feeding effect ofSEQ ID NO: 19 on the slugs was significantly (p<0.001) increased at80.7% within 6 h when compared to the water treatment (10.0%) and SEQ IDNO: 25 (16.7%), and significantly (P<0.01) increased when compared toSEQ ID NO: 26 (23.3%) (FIG. 9C, exemplary result).

While the invention has been described with reference to details of theillustrated embodiments, these details are not intended to limit thescope of the invention as defined in the appended claims. The embodimentof the invention in which exclusive property or privilege is claimed isdefined as follows:

What is claimed is:
 1. A composition comprising an isolated syntheticpeptide having SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 4,or a combination thereof in an aqueous solution at a concentration of atleast 10 nmol, and further comprising a slug food material andoptionally a phagostimulant.
 2. The composition of claim 1, comprisingSEQ ID NO:
 18. 3. The composition of claim 1, comprising SEQ ID NO: 17.4. The composition of claim 1, comprising SEQ ID NO:
 3. 5. Thecomposition of claim 1, comprising SEQ ID NO:
 4. 6. A molluscicidecomposition for controlling a slug, the molluscicide comprising aneffective amount of an isolated synthetic peptide having SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 4, or a combination thereof inan aqueous solution at a concentration of at least 10 nmol, and furthercomprising a slug food material and optionally a phagostimulant.
 7. Themolluscicide composition of claim 6, wherein the synthetic peptidecomprises SEQ ID NO:
 18. 8. The molluscicide composition of claim 6,wherein the synthetic peptide comprises SEQ ID NO:
 17. 9. Themolluscicide composition of claim 6, wherein the synthetic peptidecomprises SEQ ID NO:
 3. 10. The molluscicide composition of claim 6,wherein the synthetic peptide comprises SEQ ID NO:
 4. 11. Themolluscicide composition of claim 6, further comprising anagriculturally acceptable carrier.
 12. The molluscicide composition ofclaim 6, further comprising a preservative, a dispersant, a fungicide,an herbicide, an attractant, a phagostimulant, a bait material, or acombination thereof.
 13. A method for controlling a slug comprisingcontacting a slug or its environment with a biologically effectiveamount of a compound of claim 1, or a molluscicide of claim 6 whereinthe mortality of said slug increases.
 14. The method of claim 13,wherein the slug is Deroceras reticulatum.